M. Andrew Walker

Genetic structure and differentiation in cultivated grape, Vitis vinifera L.

222 cultivated (Vitis vinifera) and 22 wild (V. vinifera ssp. sylvestris) grape accessions were analysed for genetic diversity and differentiation at eight microsatellite loci. A total of 94 alleles were detected, with extensive polymorphism among the accessions. Multivariate relationships among accessions revealed 16 genetic groups structured into three clusters, supporting the classical eco-geographic grouping of grape cultivars: occidentalis, pontica and orientalis. French cultivars appeared to be distinct and showed close affinity to the wild progenitor, ssp. sylvestris from south-western France (Pyrenees) and Tunisia, probably reflecting the origin and domestication history of many of the old wine cultivars from France. There was appreciable level of differentiation between table and wine grape cultivars, and the Muscat types were somewhat distinct within the wine grapes. Contingency chi2 analysis indicated significant heterogeneity in allele frequencies among groups at all loci. The observed heterozygosities for different groups ranged from 0.625 to 0.9 with an overall average of 0.771. Genetic relationships among groups suggested hierarchical differentiation within cultivated grape. The gene diversity analysis indicated narrow divergence among groups and that most variation was found within groups (approximately 85%). Partitioning of diversity suggested that the remaining variation is somewhat structured hierarchically at different levels of differentiation. The overall organization of genetic diversity suggests that the germplasm of cultivated grape represents a single complex gene pool and that its structure is determined by strong artificial selection and a vegetative mode of reproduction.

The present and future of the international wine industry

Wine production is both art and science, a blend of individual creativity and innovative technology. But wine production is also business, with economic factors driving manufacturing practices. To be successful in the modern marketplace, a winemaker must integrate the artistic and economic aspects of wine production, and possess a solid understanding of the intrinsic and extrinsic factors that underlie purchase motivation.

Abundance of phytoseiid mites on Vitis species: effects of leaf hairs, domatia, prey abundance and plant phylogeny

We observed the number of predatory mites (Phytoseiidae:Typhlodromus caudiglans) on the foliage of 20 North American species of grapes (Vitis spp) plus the domesticated EuropeanVitis vinifera, all grown in a common garden. We found relatively few phytophagous mites. The numbers of phytophagous mites were not correlated with the plant characteristics that we measured. We found approximately five times as many predatory mites as phytophagous mites and the numbers of these phytoseiid predators were not affected by the availability of prey. Similarly, numbers of phytoseiids were unaffected by plant gender and, hence, the availability of pollen, another source of food. The numbers of phytoseiids were not clustered according to the taxonomic grouping of the tested plant species. Leaf surface characteristics explained over 25% of the variance in the numbers of phytoseiids. Numbers of phytoseiids were positively associated with the density of vein hairs, the density of bristles in leaf axils, and the presence of leaf domatia. These results suggest that sheltered habitats rather than food availability may limit the numbers of phytoseiid mites on grapevines.

Water uptake along the length of grapevine fine roots: developmental anatomy, tissue specific aquaporin expression, and pathways of water transport

Peak aquaporin expression/activity and hydraulic conductivity occurred in root tips and interior tissues; contrary to theoretical predictions, low aquaporin expression and activity in suberized secondary growth portions of fine roots suggests a limited role in controlling water uptake in this region of the root. To better understand water uptake patterns in root systems of woody perennial crops, we detailed the developmental anatomy and hydraulic physiology along the length of grapevine (Vitis berlandieri × Vitis rupestris) fine roots from the tip to secondary growth zones. Our characterization included the localization of suberized structures and aquaporin gene expression and the determination of hydraulic conductivity (Lpr) and aquaporin protein activity (via chemical inhibition) in different root zones under both osmotic and hydrostatic pressure gradients. Tissue-specific messenger RNA levels of the plasma membrane aquaporin isogenes (VvPIPs) were quantified using laser-capture microdissection and quantitative polymerase chain reaction. Our results highlight dramatic changes in structure and function along the length of grapevine fine roots. Although the root tip lacked suberization altogether, a suberized exodermis and endodermis developed in the maturation zone, which gave way to the secondary growth zone containing a multilayer suberized periderm. Longitudinally, VvPIP isogenes exhibited strong peaks of expression in the root tip that decreased precipitously along the root length in a pattern similar to Arabidopsis (Arabidopsis thaliana) roots. In the radial orientation, expression was always greatest in interior tissues (i.e. stele, endodermis, and/or vascular tissues) for all root zones. High Lpr and aquaporin protein activity were associated with peak VvPIP expression levels in the root tip. This suggests that aquaporins play a limited role in controlling water uptake in secondary growth zones, which contradicts existing theoretical predictions. Despite having significantly lower Lpr, woody roots can constitute the vast majority of the root system surface area in mature vines and thus provide for significant water uptake potential.

A SNP transferability survey within the genus Vitis

BackgroundEfforts to sequence the genomes of different organisms continue to increase. The DNA sequence is usually decoded for one individual and its application is for the whole species. The recent sequencing of the highly heterozygous Vitis vinifera L. cultivar Pinot Noir (clone ENTAV 115) genome gave rise to several thousand polymorphisms and offers a good model to study the transferability of its degree of polymorphism to other individuals of the same species and within the genus.ResultsThis study was performed by genotyping 137 SNPs through the SNPlex™ Genotyping System (Applied Biosystems Inc.) and by comparing the SNPlex sequencing results across 35 (of the 137) regions from 69 grape accessions. A heterozygous state transferability of 31.5% across the unrelated cultivars of V. vinifera, of 18.8% across the wild forms of V. vinifera, of 2.3% among non-vinifera Vitis species, and of 0% with Muscadinia rotundifolia was found. In addition, mean allele frequencies were used to evaluate SNP informativeness and develop useful subsets of markers.ConclusionUsing SNPlex application and corroboration from the sequencing analysis, the informativeness of SNP markers from the heterozygous grape cultivar Pinot Noir was validated in V. vinifera (including cultivars and wild forms), but had a limited application for non-vinifera Vitis species where a resequencing strategy may be preferred, knowing that homology at priming sites is sufficient. This work will allow future applications such as mapping and diversity studies, accession identification and genomic-research assisted breeding within V. vinifera.

Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator

BackgroundPowdery mildew, caused by the obligate biotrophic fungus Erysiphe necator, is an economically important disease of grapevines worldwide. Large quantities of fungicides are used for its control, accelerating the incidence of fungicide-resistance. Copy number variations (CNVs) are unbalanced changes in the structure of the genome that have been associated with complex traits. In addition to providing the first description of the large and highly repetitive genome of E. necator, this study describes the impact of genomic structural variation on fungicide resistance in Erysiphe necator.ResultsA shotgun approach was applied to sequence and assemble the genome of five E. necator isolates, and RNA-seq and comparative genomics were used to predict and annotate protein-coding genes. Our results show that the E. necator genome is exceptionally large and repetitive and suggest that transposable elements are responsible for genome expansion. Frequent structural variations were found between isolates and included copy number variation in EnCYP51, the target of the commonly used sterol demethylase inhibitor (DMI) fungicides. A panel of 89 additional E. necator isolates collected from diverse vineyard sites was screened for copy number variation in the EnCYP51 gene and for presence/absence of a point mutation (Y136F) known to result in higher fungicide tolerance. We show that an increase in EnCYP51 copy number is significantly more likely to be detected in isolates collected from fungicide-treated vineyards. Increased EnCYP51 copy numbers were detected with the Y136F allele, suggesting that an increase in copy number becomes advantageous only after the fungicide-tolerant allele is acquired. We also show that EnCYP51 copy number influences expression in a gene-dose dependent manner and correlates with fungal growth in the presence of a DMI fungicide.ConclusionsTaken together our results show that CNV can be adaptive in the development of resistance to fungicides by providing increasing quantitative protection in a gene-dosage dependent manner. The results of this work not only demonstrate the effectiveness of using genomics to dissect complex traits in organisms with very limited molecular information, but also may have broader implications for understanding genomic dynamics in response to strong selective pressure in other pathogens with similar genome architectures.

Behavioral and population consequences of acarodomatia in grapes on phytoseiid mites (Mesostigmata) and implications for plant breeding

We examined the influence of acarodomatia in the riverbank grape Vitis riparia Michaux (Vitaceae) on the distribution and abundance of predatory mites (Phytoseiidae) and their interactions with herbivorous mites. Acarodomatia are tufts of nonglandular trichomes or pits located in major leaf vein axes of many species of woody perennial plants and are often occupied by predatory and mycophagous mites. In common garden plantings of different accessions of V. riparia we found a significant positive relationship between size of domatia and the abundance of naturally occurring predatory mites. Behavior of adult predatory mites may explain this positive association, in part. In separate laboratory experiments, gravid females of Typhlodromus pyri Scheuten and Amblyseius andersoni Chant spent more time and deposited more eggs on half of a V. riparia leaf with accessible domatia versus the other half in which access to domatia was blocked with pruning tar. Domatia also had population consequences. In an outdoor experiment using potted grapevines, population size of T. pyri and A. andersoni mites was greater on V. riparia with open domatia compared to V. riparia in which domatia were blocked with pruning tar. Population size of predatory mites was also greater on V. riparia with domatia than on Vitis vinifera L., whether their axils were blocked or not. Since V. vinifera have very small domatia, these results indicate that the presence of domatia is important, not just access to vein axils. Elevated predatory mite populations in response to domatia, however, did not translate into differences in the abundance of European red mite Panonychus ulmi (Koch), an important pest of grapes.

Dormant Buds and Adventitious Root Formation by Vitis and Other Woody Plants

Viticulture has historically depended upon clonal propagation of winegrape, tablegrape, and rootstock cultivars. Dependence on clonal propagation is perpetuated by consumer preference, legal regulations, a reproductive biology that is incompatible with sustaining genetic lines, and the fact that grapevine breeding is a slow process. Adventitious root formation is a key component to successful clonal propagation. In spite of this fact, grapevine has not been a centerpiece for adventitious root research. Dormant woody canes represent complex assemblages of tissues and organs. Factors that further contribute to such complexity include levels of endogenous plant growth regulators, the extent and duration of dormancy, carbohydrate storage, transport, the presence or absence of dormant buds or emergent shoots, and preconditioning treatments. For the above reasons, the mechanisms driving adventitious root formation by grapevine and other woody cuttings are poorly understood. We present results indicating that the dormant bud on cane cuttings from a non-recalcitrant to root Vitis vinifera cultivar, cv. Cabernet Sauvignon, slows or inhibits adventitious root emergence. In contrast to Cabernet Sauvignon, removal of the dormant bud from cane cuttings of a recalcitrant to root hybrid rootstock (V. berlandieri × V. riparia cv. 420A) and an intermediate to root hybrid rootstock (V. riparia × V. rupestris cv. 101-14) had no influence on adventitious root emergence. Reciprocal transplanting of nodes containing dormant buds among all three cultivars did not affect rooting behavior. Our results indicate that the commonly held belief that bud removal diminishes adventitious root emergence is not true.

Multilocus Simple Sequence Repeat Markers for Differentiating Strains and Evaluating Genetic Diversity of Xylella fastidiosa

ABSTRACT A genome-wide search was performed to identify simple sequence repeat (SSR) loci among the available sequence databases from four strains of Xylella fastidiosa (strains causing Pierces disease, citrus variegated chlorosis, almond leaf scorch, and oleander leaf scorch). Thirty-four SSR loci were selected for SSR primer design and were validated in PCR experiments. These multilocus SSR primers, distributed across the X. fastidiosa genome, clearly differentiated and clustered X. fastidiosa strains collected from grape, almond, citrus, and oleander. They are well suited for differentiating strains and studying X. fastidiosa epidemiology and population genetics.

Scanning Electron Microscopy Reveals Different Response Pattern of Four Vitis Genotypes to Xylella fastidiosa Infection

The xylem-limited bacterium Xylella fastidiosa causes Pierces disease (PD), whose disease symptoms are primarily the result of xylem vessel blockage in susceptible grapevines. Stem internode and petiole tissues from infected and uninfected control plants of four grape genotypes (Vitis vinifera, V. rufotomentosa, V. smalliana, and V. arizonica/candicans) differing in PD susceptibility were examined using scanning electron microscopy (SEM). Tyloses, fibrillar networks, and gum plugs were observed in lumens of tracheary elements in petioles and internodes of both water-inoculated control plants and X. fastidiosa-inoculated plants of all genotypes. Bacteria were not observed in control plants. In both petiole and internode tissues, the greatest number of occluded xylem vessels were observed in V. vinifera and the smallest number in V. arizonica/candicans. The number of xylem vessels infested with X. fastidiosa was greatest in V. vinifera and did not differ among the other three genotypes. Systemic infection was found in all genotypes. The frequency with which X. fastidiosa infested vessels were observed using SEM corresponded well with bacterial levels estimated by enzyme-linked immunosorbent assay. Among infected plants, tylose formation in internodes was lowest in V. arizonica/candicans and did not differ among the other three genotypes. Infection with X. fastidiosa strongly induced tylose formation in V. vinifera and V. smalliana but not in V. arizonica/candicans. Analysis across tissues and genotypes indicated an induction of fibrillar networks and gum occlusions in response to X. fastidiosa infection, whereas treatment comparisons within genotypes were not significant except for V. vinifera petioles. Limiting the spread of X. fastidiosa infection by xylem conduit occlusions does not appear to be the mechanism conferring PD resistance or tolerance to V. arizonica/candicans, V. smalliana, or V. rufotomentosa. In contrast, the strong induction of tyloses may be detrimental rather than beneficial for V. vinifera survival after X. fastidiosa infection.